Serum vitamin D, blood pressure and hypertension risk in the HUNT study using observational and Mendelian randomization approaches

Limited studies have triangulated the relationship between serum 25-hydroxyvitamin D [25(OH)D] levels and systolic blood pressure (SBP), diastolic blood pressure (DBP) or hypertension risk utilizing both observational and Mendelian randomization (MR) approaches. We employed data from the Norwegian Trøndelag Health Study (HUNT) to conduct cross-sectional (n = 5854) and prospective (n = 3592) analyses, as well as one-sample MR (n = 86,324). We also used largest publicly available data for two-sample MR. Our cross-sectional analyses showed a 25 nmol/L increase in 25(OH)D was associated with a 1.73 mmHg decrease in SBP (95% CI − 2.46 to − 1.01), a 0.91 mmHg decrease in DBP (95% CI − 1.35 to − 0.47) and 19% lower prevalence of hypertension (OR 0.81, 95% CI 0.74 to 0.90) after adjusting for important confounders. However, these associations disappeared in prospective analyses. One-sample and two-sample MR results further suggested no causal relationship between serum vitamin D levels and blood pressure or hypertension risk in the general population.


Supplementary text 1
We assessed the third assumption for the MR analyses (test whether there existed horizontal pleiotropy) using SNP-based two-sample methods such as MR-Egger 1 , weighted median 2 and MR-Pleiotropy Residual Sum and Outlier (MR-PRESSO) 3 methods in the HUNT population.MR-Egger method estimates the causal effect allowing for the presence of pleiotropy.The intercept term in the MR-Egger regression represents the average pleiotropic effect.A non-zero intercept and the P value of the intercept test <0.05 indicates the presence of horizontal pleiotropy 1 .Weighted median method is another robust method that can provide valid casual estimates even when up to 50 % of the SNPs exhibit pleiotropic effects 2 .MR-PRESSO method is used to identify and correct for potential outliers (P<0.05) 3 .This method is based on a modified regression model that includes a correction term for pleiotropy based on a residual-based approach.Since each method is robust to different patterns of horizontal pleiotropy, a good agreement across all the methods suggests that pleiotropy would not influence the causal estimate.

Supplementary text 3
To test whether there existed non-linear causal associations in the MR analyses, both the residual method and the doubly-ranked method were used in the sub-cohort of the HUNT population (n=5854).The residual method is a widely used method for the estimation of localized average causal effects at different levels of the exposure distribution 9 .We used the residuals of serum 25(OH)D to divide the sub-cohort of HUNT population into four equal-sized strata under the assumption that the genetic effect on serum 25(OH)D is constant within each stratum 9,10 .The residual of serum 25(OH)D was calculated as the residual from regression of serum 25(OH)D on the mean-centred genetic risk score 10 .The advantage to use residual of serum 25(OH) D rather than the actual serum 25(OH)D levels to stratify the population is to avoid collider bias 10 .The doubly-ranked method is a non-parametric stratification method 11 .In the doubly-ranked method, the population from the sub-cohort of HUNT Study was divided into four equal-sized strata based on rank of the externally weighted genetic risk score from lowest to highest and then the exposure in each stratum was ranked from lowest to highest levels.This method may give unbiased estimates even when the constant genetic effect assumption is violated 11 .However, in a recent paper by Hamilton et al. 12 , it suggested that both methods might have provided biased estimates using age and sex as negative control outcomes within the UK Biobank data.The authors suggested that the biased results with the two non-linear methods might be due to selection bias in the UK Biobank data, or population stratification or unclear problems in the methods.Hence, the observed inverse association between serum 25(OH)D and SBP within the lowest stratum using both methods in our study might also be due to unknown bias.

Variables
Population with information on genetic data 2 Significance of Bonferroni corrected P value was calculated as 0.05/8=0.006. 3Active: physical activity level from low to high. 3 Inactive: no physical activity or only light physical activity ≤2h per week.
Supplementary Table 3. Sensitivity analyses to address potential horizontal pleiotropy for the causal associations of serum 25(OH)D levels (per 25 nmol/L increase) with systolic blood pressure, diastolic blood pressure and risk of hypertension in the HUNT study 1  1 SNP-based two sample methods were applied to a sub-cohort consisting of 5854 individuals to investigate the gene-exposure association, while the analysis for gene-outcome associations was performing in the total cohort of 86,324 individuals in the HUNT study. 2 Coefficient was derived from linear regression for SBP/DBP as outcomes while OR was derived from logistic regression for hypertension as outcome in MR-Egger, weighted median and MR-PRESSO methods.All results corresponded to per 25 nmol/L increase in genetically determined serum 25(OH)D after adjustment for age, sex, batch and 20 PCs. 3 The results based on the MR-PRESSO method were presented only if outliers were detected.For the associations between serum 25(OH)D levels and systolic blood pressure or diastolic blood pressure, no outliers were detected., the UK Biobank GWAS for SBP and DBP (n=757,601) by Evengelou et al. 7 , and the GWAS for hypertension from FinnGen, which included 42,857 cases and 218,792 controls 8 .The datasets for the exposure and the outcomes were harmonized for each analysis. 2.In these two-sample MRs, we employed the same summary data for gene to outcome associations as in a prior two-sample MR, which used 6 SNPs from Jiang et al.

Supplementary
as instruments for serum 25(OH)D levels: the UK Biobank GWAS for SBP and DBP (n=757,601) by Evengelou et al. 7 , and the GWAS for hypertension from FinnGen, which included 42,857 cases and 218,792 controls 8 .The datasets for the exposure and the outcomes were harmonized for each analysis. 2Coefficient was derived from linear regression for SBP and DBP as outcomes while OR was derived from logistic regression for hypertension as outcome corresponding to 1 standard deviation increase in genetically determined log-transformed serum 25(OH)D in inverse-variance weighted, MR-Egger and weighted median methods. .

Table 2 .
The associations between externally weighted genetic risk score of serum 25(OH)D and potential confounders in the sub-cohort of the HUNT Study (n=5854)

Table 4 .
Two-sample MR¹ results for the causal associations of serum 25(OH)D levels with systolic blood pressure, diastolic blood pressure and risk of hypertension using 6 SNPs from Jiang et al. as instruments for serum 25(OH)D levels CI: Confidence interval; Coef: Coefficient; DBP: Diastolic blood pressure; MR: Mendelian randomization; OR: Odds ratio; SBP: Systolic blood pressure; SNP: Single Nucleotide Polymorphism; 25(OH)D: 25-hydroxyvitamin D 1 Two-sample MR was performed with summary data from different genome-wide association studies (GWAS) sources: the Underlying Genetic Determinants of Vitamin D and Highly Related Traits (SUNLIGHT) Consortium for serum 25(OH)D (n=79,366) by Jiang et al.

Table 5 .
5wo-sample MR¹ results for the causal associations of serum 25(OH)D levels with systolic blood pressure, diastolic blood pressure and risk of hypertension using two sets of instruments selected from recent large GWASs for serum 25(OH)D levels Additional two-sample MR studies were performed using another two sets of instruments selected from recent large GWASs for serum 25(OH)D levels: 35 single nucleotide polymorphism (SNP)s from the study by Zhou et al. (n=294,970)5and 69 SNPs from the study byManousaki et al. (n=443,734) 1

Table 7 .
Characteristics of 19 SNPs included in the externally weighted genetic risk score for serum 25(OH)D in the HUNT Study (n=86,324) Supplementary

Table 8 .
Characteristics of SNPs used as genetic instruments for serum 25(OH)D levels from different GWASs in two-sample MR Chromosome number; MR: Mendelian randomization; GWAS: Genome-wide association study; Position_b37: Base pair position from Genome Reference Consortium Human Build (GRCh37); SE: Standard error; SNP: Single Nucleotide Polymorphism; 25(OH)D: 25-hydrovitamin D